Whole cell selection utilizing azlactone-functional supports

ABSTRACT

A method for cell selection is disclosed. The method uses an azlactone-functional support, which is derivatized with and covalently coupled to a substance that is biologically active with a desired type of whole cell. The method allows the whole cells in the mixture to interact with and bind to the coupled biologically active substance, after which a remainder of the mixture is removed from the support. Optionally, one can elute the bound whole cells from the coupled biologically active substance to produce a purified collection of the whole cells.

FIELD OF INVENTION

[0001] This invention relates to the use of azlactone-functionalsupports to provide cell selection.

BACKGROUND OF INVENTION

[0002] Rapidly expanding knowledge in the areas of molecular andcellular biology, immunology, and genetics has led to the identificationand characterization of a variety of highly specialized subpopulationsof cells within many types of tissues. For example, the identificationof rare “stem cells” in such crucial tissues as the brain, islet cellsof the pancreas, and liver have led to speculation that one day manydiseased tissues may be treated by regeneration of healthy tissuefollowing cell transplantation (Beardsley, Scientific American, June1998, pp. 11-12). Such therapies have, in fact, already been achieved inareas such as cancer treatment. High doses of chemotherapy or radiationneeded to destroy cancerous tissue often also destroy the patient's bonemarrow and, effectively, his entire immune system. A transplant ofhematopoietic stem cells, previously isolated from either marrow orperipheral blood, can rescue the patient by reconstituting the bonemarrow and cells of the immune system (Donahue, et al., Blood, 1996, 87,pp. 1644-1653). Purified stem cells or other specific cell populationsare also believed to be important for developing a variety ofimmunotherapies (e.g., AIDS treatments) and gene therapy.

[0003] To fuel research, and indeed clinical applications, in these andrelated areas, increasingly effective methods of separation andpurification of various cell populations are required. Over the years, avariety of methods of cell separation/purification have been utilized.These separation techniques have depended upon various biological andbiochemical, physical, or immunological characteristics of the cellpopulation to be separated (Esser, in “Cell Separation Methods andApplications”, D. Recktenwald and A. Radbruch, Eds., Marcel Dekker, NY,N.Y., 1997, pp. 1-14). Immunoaffinity-based separations have shownconsiderable promise in terms of providing relatively pure preparationsof specific cells. U.S. Pat. No. 5,035,994 (Civin) describes the use ofa solid-phase linked monoclonal antibody which binds specifically to anantigen on human pluripotent lympho-hematopoietic stem cells to separatesaid stem cells from a suspension of marrow or blood cells. Pope, etal., (Bioconjugate Chem., 1993, 4, pp. 166-171) describe the use ofbifunctional silane reagents to activate glass and cellulose solidsupports. Goat anti-mouse antibodies are then covalently linked to theactivated supports and the derivatized supports used to selectivelydeplete CD34+ or CD4+ mononuclear cells from peripheral blood samples.U.S. Pat. No. 5,215,927 (Berenson, et al.) describes the immunoselectionof cells using an avidin-biotin recognition system. While these andother methods described in the art allow for selection and purificationof selected cell populations, there is a continued need for new methodsand materials that can provide those cell populations in improvedpurities and yields.

[0004] Azlactone-functional supports have been described to be quiteuseful for the immobilization of biologically active materials. Forexample, U.S. Pat. No. 5,403,902 (Heilmann, et al.) describes thepreparation of particulate or beaded materials to whichbiomacromolecules such as proteins, antibodies, enzymes, etc. can becoupled. These materials are useful, for example, in the affinitychromatographic purification of proteins. U.S. Pat. Nos. 5,262,484,5,292,514, 5,451,453, 5,486,358, and 5,510,421 all describe otherazlactone-functional supports and materials and their uses. In none ofthese references has it been described or suggested thatazlactone-functional supports might be useful for whole cellpurification or selection.

[0005] PCT Patent Publication WO 93/04576 (Kim et al.) describes theability of azlactone-functional supports to separate proteinaceousmaterials from nonproteinaceous materials. This can be useful forseparation and purification of biological materials. When theazlactone-functional support is contacted with a mixture ofproteinaceous and nonproteinaceous materials, the proteinaceousmaterials react with and become coupled to the support, and thenonproteinaceous materials (e.g., nucleic acids) do not react with thesupport, but remain in solution. This publication does not describe theability to separate whole cells from naturally-occurring biologicalfluids, but does disclose that the nonproteinaceous material retainsbiological activity for further processing after separation from theazlactone-functional support that has proteinaceous material coupledthereto.

[0006] PCT Patent Publication WO 94/22918 (Velander et al.) describes amethod of derivatizing a porous support in a manner that distributes theligand. Examples disclosed in the publication identify the covalentcoupling of monoclonal antibodies and proteins for further biologicalseparation processes.

[0007] U.S. Pat. No. 5,200,471 (Coleman et al.) describes a method ofcovalently coupling ligands to azlactone-functional supports,particularly with a method to increase the quality of the covalentlycoupled ligands, to retain high specific bound biological activity.

[0008] U.S. Pat. No. 5,561,097 (Gleason et al.) describes a method ofcovalently coupling small molecule ligands to azlactone-functionalsupports in a manner that can control the density and distribution ofthe ligands. This method is useful for the preparation ofchromatographic supports.

[0009] Summary of Invention

[0010] A need exists for new materials and methods for the selection orpurification of whole cells. It has now been found thatazlactone-functional support materials, previously known to be usefulfor preparation of chromatographic supports for protein purification orfor the preparation of covalently coupled ligands such as proteins,enzymes, and the like, can also serve as starting materials for thepreparation of supports for whole cell selection and purification.

[0011] Briefly, one aspect of the invention provides a method for cellselection comprising the steps of (a) providing an azlactone-functionalsupport, (b) derivatizing the azlactone-functional support with asubstance that is biologically active towards a desired type of wholecell, wherein the substance is covalently coupled to theazlactone-functional support, (c) contacting the product of step (b)with a mixture containing the whole cells, (d) allowing the whole cellsin the mixture to interact with and bind to the coupled biologicallyactive substance, (e) removing a remainder of the mixture from thesupport, and (f) optionally, eluting the bound cells from the coupledbiologically active substance to produce a purified collection of thewhole cells.

[0012] “Support” means any article that is or can be madeazlactone-functional. Acceptable supports for use in the presentinvention can vary widely within the scope of the invention. A supportcan be porous or nonporous, depending on preferred final use. A supportcan be continuous or non-continuous depending on ultimate desired usage.A support can be made of a variety of materials, including supports madeof ceramic, glassy, metallic, or polymeric materials or combinations ofmaterials. A support can be flexible or inflexible depending on ultimatedesired usage.

[0013] “Azlactone-functional” means that a support hasazlactone-functional groups on internal and/or external surfaces of suchsupport. Thus, such reactive supports have an azlactone-functional groupof the formula:

[0014] wherein:

[0015] R¹ and R² independently can be an alkyl group having 1 to 14carbon atoms, a cycloalkyl group having 3 to 14 carbon atoms, an arylgroup having 5 to 12 ring atoms, an arenyl group having 6 to 26 carbonatoms and 0 to 3 S, N, and nonperoxidic 0 heteroatoms, or R¹ and R²taken together with the carbon to which they are joined can form acarbocyclic ring containing 4 to 12 ring atoms, and n is an integer 0 or1.

[0016] “Covalently coupled” means chemically attached by means of acovalent bond.

[0017] “Biologically active substance” means substances which, oncecovalently coupled with and immobilized on the azlactone-functionalsupport, are useful in providing selective or specific interaction withcertain target whole cell populations.

[0018] “Whole cell” means a biologically active plant or animal cellthat retains its structure intact during separation from otherbiological materials and is capable of remaining biologically activeafter use of the aziactone-functional support having a biologicallyactive substance covalently coupled thereto to separate such plant oranimal cell from other biological materials. Azlactone-functionalsupports useful for the preparation of cell selection supports includebeaded or particulate supports such as those disclosed in U.S. Pat. No.5,403,902, porous supports such as those disclosed in U.S. Pat. No.5,344,701 and in PCT Patent Publication WO 93/06925, membrane supportssuch as those disclosed in U.S. Pat. No. 5,510,421, blends and articlesprepared therefrom such as those disclosed in U.S. Pat. No. 5,408,002,substrates such as those disclosed in U.S. Pat. No. 5,292,514, and graftcopolymers and articles prepared therefrom such as those disclosed inU.S. Pat. Nos. 5,013,795 and 5,262,484. All of these patents andpublications are incorporated by reference herein.

[0019] The biologically active substance used to derivatize theazlactone-functional support via covalent coupling can interact directlywith the specific whole cell population intended to be selected.Nonlimiting examples of such substances include an antibody (Ab)directed toward a specific cell surface marker (or antigen, Ag)expressed on the surface of the whole cell to be selected.Alternatively, the biologically active substance may interact with theselected whole cell population through a second, intermediarybiologically active substance.

[0020] A feature of the present invention is the ease of preparation ofsupports useful for whole cell selection.

[0021] Another feature of the present invention is the versatility andvariety of methods available for the preparation of azlactone-functionalsupports that can be modified to be useful for whole cell selection.

[0022] Another feature of the present invention is that the surfacecharacteristics of the whole cell selection supports can be readilycontrolled to minimize or prevent nonspecific interactions withnon-target whole cells.

[0023] An advantage of the invention is that target whole cellpopulations may be isolated which have higher purities than thoseisolated using conventional supports, such as those identified above inthe Background of the Invention that utilize chemistries other thanazlactone chemistry.

[0024] Another advantage of the present invention is that target wholecell populations may be isolated in higher yields than those isolatedusing conventional supports and in a manner that retains biologicallyactivity.

[0025] Further features and advantages are disclosed in the followingembodiments of the invention.

[0026] Embodiments of Invention

[0027] Azlactone-Functional Supports

[0028] Azlactone-functional supports can be any compound or materialcontaining or comprising at least one azlactone moiety that can bederivatized by covalent reaction with a biologically active substance.Such azlactone-functional supports are well known in the art.Preferably, the azlactone-functional support is a solid, insolublematerial, wherein the term “insoluble” means does not dissolve in themedium from which the whole cell selection is to take place, comprisingazlactone moieties on its surface which are readily available forreaction with the biologically active substance useful for whole cellselection.

[0029] Nonlimiting examples of azlactone-functional supports includebeads, particulates, membranes, woven and nonwoven webs, and solidplastic articles comprising azlactone moieties on their surfaces. Suchtypes of azlactone-functional supports are variously disclosed in thecollection of U.S. Patents incorporated by reference above, all of whichare owned by Minnesota Mining and Manufacturing Company (3M) of St.Paul, Minn., USA.

[0030] More particularly, beaded and particulate azlactone-functionalsupports are extensively described in U.S. Pat. No. 5,403,902 (Heilmann,et al.) incorporated by reference herein. These supports are prepared byreverse phase suspension polymerization processes and by dispersionpolymerization processes from 2-alkenyl azlactone monomers and,optionally, comonomers and crosslinkers.

[0031] Porous azlactone-functional supports, such as membranes andnonwoven materials, are described in U.S. Pat. No. 5,344,701 (Gagnon, etal.) incorporated by reference herein. These supports are prepared bygraft polymerization of azlactone monomers and, optionally, comonomersto the surfaces of preexisting supports using high-energy radiation.Alternatively, aziactone monomers, crosslinkers, and optionallycomonomers are coated on the surfaces of the preexisting supports, thenpolymerized to produce the azlactone-functional supports.

[0032] Other porous azlactone-functional supports are described in PCTPatent Publication WO 93/06925 (Rasmussen, et al.) and copending,coassigned, U.S. patent application Ser. No. 08/776,601, incorporatedherein by reference, in which azlactone-functional particles areincorporated into a continuous porous matrix such as a fibrillatedpolytetrafluoroethylene membrane or a nonwoven web.

[0033] Azlactone-functional membranes prepared by solvent phaseinversion techniques are described in U.S. Pat. No. 5,510,421 (Dennison,et al.) incorporated by reference herein.

[0034] Thermoplastic azlactone-functional graft copolymers and copolymerblends are described in U.S. Pat. Nos. 5,013,795; 5,262,484; and5,408,002 (all Coleman, et al.) incorporated herein by reference. Thesecompositions are useful for the preparation of azlactone-functionalmolded plastic articles such as microtitration wells and plates, petridishes, tubing, body implants, test tubes, centrifuge tubes, beakers,cuvettes, etc., which are also useful as substrates for the preparationof supports for whole cell selection in accordance with this invention.

[0035] Other azlactone-functional substrates useful in this inventionare those disclosed in U.S. Pat. No. 5,292,514 (Capecchi, et al.)incorporated by reference herein.

[0036] Biologically Active Substances

[0037] Biologically active substances useful for the present inventioncan interact with the target whole cells either directly or indirectly,i.e. through the intermediacy of one or more secondary biologicallyactive substances.

[0038] Biologically active substances capable of direct interaction withtarget whole cells include, but are not limited to, antibodies to wholecell surface antigens, lectins, and other proteins known to interactwith whole cell surfaces. A wide variety of specific whole cell surfaceantigens have been identified, commonly referred to as CD antigens(“Cell Separation Methods and Applications”, D. Recktenwald and A.Radbruch, Eds., Marcel Dekker, NY, N.Y., 1997, pp. 297-319). It is wellwithin the capability of one skilled in the art to prepare antibodies tothese antigens. In fact, many such antibodies are currently availablefrom commercial sources such as R&D Systems, Minneapolis, Minn., andBoehringer Mannheim Corp., Indianapolis, Ind. Covalent coupling of theseantibodies to aziactone-functional supports produces supports capable ofdirect interaction and selection of target whole cells viaantibody-antigen bonding interactions.

[0039] Other biologically active substances immobilized onaziactone-functional supports can interact with target whole cellsindirectly. For example, it is within the scope of the invention to usean intermediary biologically active substance which is minimally“bifunctional”, i.e. it has functionality which exhibits a specificrecognition or interaction with the target whole cell population butalso has a second functionality which interacts and bonds to thebiologically active substance which is coupled to theazlactone-functional support. An example of such an intermediarysubstance would be an antibody to a whole cell surface antigenconjugated to an antigen that specifically interacts with a secondantibody immobilized on the support. Another example, and a preferredintermediary for use in this invention, is an anti-CD34+ antibodyconjugated to biotin. Whole cell selection is accomplished viainteraction of the antibody portion with the target whole cell and ofbiotin with avidin immobilized on the support.

[0040] Methods of Derivatizing Supports

[0041] Methods for the immobilization of biologically active substanceson azlactone-functional supports are well known in the art, and aredescribed in detail in the above mentioned patent references. Improvedimmobilization conditions for proteins and antibodies, specifically, aretaught in U.S. Pat. No. 5,200,471 (Coleman, et al.) and in PCT PatentPublication WO 94/22918 (Velander, et al.) and companion copending,coassigned, U.S. patent application Ser. No.: 08/296,588 (allincorporated herein by reference). Additionally, U.S. Pat. No. 5,561,097(Gleason, et al.), incorporated by reference, describes techniques forcontrolling density of ligands coupled to azlactone-functional supports.

[0042] Usefulness of the Invention

[0043] The versatility and simplicity of immobilization conditions,together with the variety of techniques available for the preparation ofazlactone-functional supports, allows the preparation and optimizationof whole cell selection supports to a degree previously unknown. Forexample, once a whole cell mixture has been identified from which atarget whole cell population is desired to be selected (e.g., bonemarrow or peripheral blood), a base polymer support can be identifiedwhich exhibits minimal nonspecific binding of whole cells from thatmixture. This ensures a high degree of purity in the selected whole cellpopulation. Next, an azlactone moiety can be incorporated into the basepolymer support by a variety of techniques disclosed in the patentreferences above to provide the active chemistry needed to covalentlycouple the biologically active substance to the support. Finally, anappropriate biologically active substance can be immobilized on thesupport to provide the whole cell selection support. Depending on theidentity of the biologically active substance and the specificcharacteristics of the target whole cells, the amount of azlactonefunctionality and the amount of biologically active substanceincorporated can be easily optimized by one skilled in the art withinroutine experimentation to produce the best whole cell selection. Inaddition, because of the unique characteristics of azlactone couplingchemistry, the surface characteristics of the final whole cell selectionsupport can be manipulated during the immobilization process to furtheroptimize the whole cell selection properties of said support. In manyinstances, whole cell selection supports can be prepared which provideselected whole cell populations with improved purities and separationyields as compared to those obtained using conventional supportsemploying polyacrylamide or polystyrene polymers or conventional bindingchemistries such as cyanogen bromide or carbodiimide-based coupling.

[0044] Further advantages of this invention are illustrated by thefollowing examples, but the particular materials and amounts thereofrecited in these examples, as well as other conditions and details,should not be construed to unduly limit this invention.

EXAMPLES

[0045] Test Methods

[0046] Whole cell selection/purification: Evaluation of the interactionof whole cells with particulate supports in the following examples wasconducted using the Ceprate™ LC Avidin Column Kit (CellPro, Inc.,Bothell, Wash.) according to the manufacturer's instructions. Selectionfor CD34+ whole cells was accomplished using an anti-human CD34biotinylated monoclonal antibody (mouse) (CellPro, Inc).

[0047] Avidin coupling densities: The density of avidin coupled toaziactone supports was evaluated utilizing a biotin-p-nitrophenyl esterreagent and protocol described by Hermanson, et al., AmericanBiotechnology Laboratory, September 1994, pp. 86-88.

Example 1 Evaluation of Nonspecific Binding with Ficolled Buffy Coat

[0048] Crosslinked supports were prepared according to the teachings ofU.S. Pat. No. 5,403,902, by reverse-phase suspension polymerization,using the polymeric stabilizer of Example 24E of that reference.Methylenebisacrylamide (MBA) was used as crosslinker, and comonomersused were dimethylacrylamide (DMAm), acrylamide (Am),vinyldimethylazlactone (VDM), or N-acryloyl-2-methylalanine (sodiumsalt) (AMA). These particulate supports were elutriated to remove smallparticles, then packed into the Ceprate LC column for evaluation. Eachcolumn was challenged with a minimum of 50×10⁶ whole cells of ficolledbuffy coat, derived from fresh whole blood; the flow-through fractionswere collected. Whole cell counts, for both lymphocytes and monocytes,were measured by fluorescence-activated cell sorting (FACS) and comparedto those of the starting sample. Table 1 lists supports evaluated, wholecell recoveries, and nonspecific binding characteristics of theavidinated polyacrylamide support provided with the commercial kit.TABLE 1 Supports Evaluated and % Cells Recovered % % Sample % W/WMonomers Lymphocytes Monocytes 1a 12.5:87.5 MBA/DMAm 74 67 1b 25:75MBA/DMAm 97 87 1c 50:50 MBA/DMAm 93 83 1d 100 MBA 71 41 1e 10:20:70MBA/VDM (hydro- 95 68 lyzed)/DMAm 1f 10:20:70 MBA/AMA/DM 25 17 Am 1g10:20:70 MBA/VDM (hydro- 93 52 lyzed)/Am 1h 10:20:70 MBA/AMA/Am 79 46Control [commercial polyacrylamide 91 81 support (MBA/Am copolymer)]

[0049] Discussion: This example indicates that several monomercombinations exhibit low nonspecific binding characteristics towardslymphocytes, which would be an important characteristic for a supportdesigned for whole stem cell selection/purification. Poorer recovery ofmonocytes is not a concern, and may in fact be an advantage over thecommercial support, since monocyte populations include T-cells whichprovide antibody-mediated immunity which one would like to minimize inthe preparation of cell-mediated immune cells, such as pluripotent stemcells. Additional comment need be made concerning samples 1e/1f and1g/1h. VDM (hydrolyzed) and AMA have the identical chemical formula,however were incorporated into the resultant polymer pairs by differentprocesses.

[0050] In the case of 1e, for example, the polymer was prepared byProcess 11 of above cited U.S. Pat. No. 5,403,902 in which VDM monomerwas copolymerized, then the resultant support was hydrolyzed to producethe test sample. Sample 1f, on the other hand, was prepared bycopolymerization of AMA, the synthetic precursor of VDM. Thus, themethod of preparation of a polymer can influence its interaction withbiological species, even though it presumably has the same chemicalformulation.

Example 2 Evaluation of Nonspecific Binding from Mobilized PeripheralBlood

[0051] The best performing supports from Example 1, as well as support1f, were evaluated as in Example 1 except that the challenge wasmobilized peripheral blood to which the anti-CD34+ antibody had beenadded prior to addition to the column. Results are listed in Table 2.TABLE 2 Cell Recovery from Mobilized Peripheral Blood % Overall CellSample % Lymphocytes % Monocytes Recovery 1a 87 100  89 1b 98 88 93 1c91 83 87 1d 62 29 52 1e 91 90 88 control 90 85 87

[0052] This example also illustrates that it is possible to find polymerbackbones that exhibit equivalent or lower nonspecific binding ascompared to the prior art.

Example 3 Azlactone/Dimethylacrylamide Supports

[0053] Particulate supports were prepared from MBA, VDM, and DMAm byreverse phase suspension polymerizations according to Process II of U.S.Pat. No. 5,403,902. In these experiments, total weight of monomers was20 g. The MBA and DMAm monomers were dissolved in a mixture of methanol(30 ml) and water (70 ml). After dissolution, sodium persulfate (0.5 g)was added and allowed to dissolve. This solution was then added to astirring solution, under nitrogen, of VDM, toluene (132 ml), heptane(243 ml), and stabilizer (0.33 g) pre-equilibrated to 35° C. Thenitrogen purge was continued for 5 minutes, thentetramethylethylenediamine (TMEDA, 0.5 ml) was added to initiate thepolymerization. Polymerization was continued for 2 hours, the productwas collected by filtration, washed with acetone, elutriated usingmethanol as liquid phase to remove particles smaller than 100 microns indiameter, and dried under vacuum to constant weight. Avidin coupling:Avidin was coupled to the supports using a solution of avidin (1 mg/ml)in 0.1 M sodium carbonate/1.4 M sodium sulfate buffer, pH 9.5. Aftercoupling, excess azlactone functionality was quenched using 0.2 Mglycine, pH 9.0. Each support was evaluated in duplicate for selectionof CD34+ whole cells. Each Ceprate LC column of support (2.0 ml)received a total of 2.0×10⁸ whole cells from a fresh marrow sample.Table 3 lists support composition, number and % yield of target wholecells selected (averages of duplicates). TABLE 3 Whole cell Selectionwith VDM/DMAm Supports Sample Composition CD34+ Whole Cells % Yield 3a10:20:70 MBA/VDM/DMAm 0.59 × 10⁶ 32.6 3b 20:20:60 MBA/VDM/DMAm 1.01 ×10⁶ 55.7 3c 47:6:47 MBA/VDM/DMAm 1.24 × 10⁶ 68.7 control 1.05 × 10⁶ 58.4

Example 4 Azlactone/Acrylamide Supports

[0054] Supports were prepared as in Example 3 substituting acrylamidefor dimethylacrylamide, and were evaluated as in Example 3. Results arelisted in Table 4.%Yields are shown relative to that of the control forCD34+ whole cells. TABLE 4 Whole cell Selection with VDM/Am SupportsSample Composition Avidin Density (μg/ml) % Yield 4a 5:20:75 MBA/VDM/Am45  94 4b 10:20:70 MBA/VDM/Am 66 120 4c 20:20:60 MBA/VDM/Am 59 101control 1000  100

[0055] Discussion: Examples 3 and 4 illustrate that excellent whole cellselection can be obtained with azlactone-based supports.

Example 5 Avidin Density Study

[0056] A 20:20:60 MBA/VDM/DMAm support similar to Example 3b wasprepared. Samples of support were challenged with varying concentrationsof avidin in order to provide a range of avidin coupling densities.Supports were evaluated as in Example 3; results are shown in Table 5,with %yield reported as relative to the commercial control. TABLE 5Whole cell Selection vs. Avidin Density Sample Avidin Density (μg/ml) %Yield 5a  80 39 5b 101 48 5c 190 90 5d 775 96 5e 1414  112  control1000  100 

[0057] These data, along with those of Example 4, indicate that supportsprepared from aziactone-functional particles perform comparably toconventional supports, but can do so at much lower avidin densitylevels.

Example 6

[0058] A 20:20:60 MBA/VDM/DMAm support was prepared by the two-stepprocedure of Process I of U.S. Pat. No. 5,403,902. After drying, thissupport was classified using a Sonic Sifter (ATM Corporation, Milwaukee,Wis.) to provide two size cuts, 8a (106-150 microns) and 8b (75-106microns). Upon hydration, sample 8a measured 204 microns averagediameter while 8b measured 155 microns. Avidin was coupled using 0.1 Mcarbonate buffer, pH 9.5 under a variety of conditions, then thesupports were evaluated for CD34+ selection using marrow and mobilizedperipheral blood samples. Coupling conditions: (a) 1 mg/ml avidin, 1.4 Msulfate; (b) 1 mg/ml avidin, no sulfate; (c) 4 mg/ml avidin, 1.4 Msulfate; (d) 3 mg/ml avidin, no sulfate. Results are listed in Table 6,with % purities and yields normalized to those of the control support.TABLE 6 Whole cell Selection with VDM/DMAm Supports Prepared by ProcessI Coupling Avidin Density Sample Condition (μg/ml) % Yield % PurityTested with Mobilized Peripheral Blood 6a a  93  94 67 6a b 1282  71 666b b 1396 115 92 6a c  126  98 73 6a d 1432  95 91 6b d 1454 110 100 Tested with Marrow 6a c  126 115 100  6a d 1432 113 106  6b d 1454 121108 

[0059] This example produced a number of surprising results: (1) Thedensity of coupled avidin was found to be much higher in the absence ofsulfate than in its presence, a result which was also verified with thesupports above prepared according to Process II. This is in sharpcontrast to prior art that showed increased protein couplingefficiencies to azlactone-functional supports in the presence of sulfate(U.S. Pat. No. 5,200,471 (Coleman et al.)). (2) Process I supportsexhibited improved % yield performance over Process II beads, despitebeing of the same chemical composition (see Example 4c). (3) Process Ibeads also exhibited exceptional purities as compared to the controlsupport. (4) Optimum performance with azlactone-functional supportsoccurs with a much smaller-particle than with the control support, a250-micron bead.

Example 7

[0060] The support synthesis described in Example 6 was repeated exceptthat polymerization was conducted using sodium persulfate as the onlyinitiator and the reaction temperature was 55° C. The classified sampleexhibited a hydrated mean size of 214 microns. Avidin was coupled at adensity of 307 μg/ml and evaluated as before, giving comparable yieldsand improved purities as compared to the control support.

Example 8

[0061] A support was prepared as in Example 7, and coupled toNeutraAvidin™ (Pierce Chemical, Rockford, Ill.) at a density of 70μg/ml. When evaluated for CD34+ selection from marrow, this supportexhibited 108% purity and 129% yield as compared to the control support.

Example 9

[0062] Microtitration plates having azlactone groups on the interiorsurfaces of the wells are molded from (a) thepolymethylmethacrylate/polyVDM blend prepared according to Example 28 ofU.S. Pat. 5,408,002 and (b) the VDM-grafted polypropylene preparedaccording to Example 44 of U.S. Pat. No. 5,262,484. Using the protocolfor whole cell “panning” in Esser, in “Cell Separation Methods andApplications”, D. Recktenwald and A. Radbruch, Eds., Marcel Dekker, NY,N.Y., 1997, p. 8, excellent whole cell selection is achieved as comparedto that using non-azlactone functional control plates.

Example 10

[0063] An azlactone-functional nonwoven pad was prepared according toExample 20 of U.S. Pat. 5,344,701. Derivatization with an anti-CD4antibody as described in Pope, et al., Bioconiugate Chem., 1993, 4, pp.166-171 allows facile depletion of CD4+ mononuclear whole cells from aperipheral blood sample in a simple flow-through filtration mode. Theporosity of the nonwoven allows unbound whole cells to easily flowthrough the construction, while the immobilized antibody captures theCD4+ whole cells.

[0064] Alternatively, azlactone-functional membranes can be madeaccording to U.S. Pat. No. 5,510,421, derivatized with an appropriateantibody, and used for whole cell selection in a crossflow filtrationmode.

Example 11 The use of Azlactone-Functional Beads to Type Human BloodCells.

[0065] Commercially available anti-sera selective for blood groups A andB is incubated with Protein A-derivatized azlactone beads (preparedaccording to Example 4 of U.S. Pat. No. 5,200,471) in phosphate-bufferedsaline (PBS) at a ratio of about 5 mg of serum IgG to 1 mL of packedbeads by rocking at ambient temperature for about 15 minutes. Unboundprotein is removed by multiple rinsing steps using PBS andcentrifugation. A sample of human blood is diluted with 13BS ten-fold.200 μL samples of the diluted blood are incubated with about 50 μL eachof the above anti-sera-Protein A-azlactone bead preparation at ambienttemperature with gentle mixing for about 15 min. The beads are allowedto settle for several minutes, then the turbidity of the supernatantliquid is compared with that of controls in which the Protein A beadswere incubated with pre-immune serum. The control samples are turbidbecause no blood cells settle with the beads to the bottom of the tube.The tubes containing cells of the same blood type as the anti-serum areclear or greatly reduced in turbidity compared with the controls.

Example 12 Typing Human Blood Through Direct Immobilization of aSpecific Antibody.

[0066] Antibodies to human blood group antigens or any other cellsurface marker may be immobilized directly onto azlactone-functionalsupports and used for blood typing (as described in Example 11 or forpurification of a particular fraction of whole cells. To remove thespecifically bound whole cells incubate the specific antigen with thebead-antibody-whole cell suspension after washing away the unbound wholecells with several rinses of isotonic buffer. For example, for releaseof selected whole cells of blood group A, dilute the suspension toapproximately 0.1 mL of beads per mL of suspension containing 1 mg/mL orgreater concentration of the required antigen. For example, blood groupA positive whole cells may be eluted using a short polysaccharideterminating in alpha-N-Ac-galactosamine. Other antigens would need to bespecific to the expressed markers on the captured whole cell surface.

Example 13 Direct Immobilization of Whole Cells to Select Specific WholeCells that Bind to Them.

[0067] Activated T-cell lymphocytes presenting cell surface marker CD-28can be purified from a population of lymphocytes (e.g., a buffy coatfraction from a whole blood sample) by their ability to bind toantigen-presenting cells or macrophages. Purified antigen presentingcells can be immobilized directly to azlactone-functional supports byincubation (with gentle agitation for about 15 minutes) of about 1million whole cells with 1-2 mL of beads in an isotonic buffer that issubstantially free of primary amine. Uncoupled whole cells are removedby settling the suspension, removing the supernatant solution, andseveral rinses. 1 mL of the immobilized antigen presenting cellpreparation is incubated with about 10 mL of a buffy coat preparationthat has been diluted with PBS to about 0.5 million whole cells/mL for30 min with gentle rocking. The support is allowed to settle, thesupernatant solution is removed, then two wash steps with five volumesof PBS are conducted. Purified T-whole cells may be removed by gentlestirring of the bead slurry while flowing PBS through the slurry (suchas by using the Ceprate device).

[0068] The invention is not limited by the embodiments described above.The following claims are made.

What is claimed is:
 1. A method for cell selection comprising the stepsof: (a) providing an aziactone-functional support, (b) derivatizing theazlactone-functional support with a substance that is biologicallyactive with a desired type of whole cell, wherein the substance iscovalently coupled to the azlactone-functional support, (c) contactingthe product of step (b) with a mixture containing the whole cells, (d)allowing the whole cells in the mixture to interact with and bind to thecoupled biologically active substance, (e) removing a remainder of themixture from the support, and (i) optionally, eluting the bound cellsfrom the coupled biologically active substance to produce a purifiedcollection of the whole cells.
 2. The method of Claim 1, wherein theazlactone-functional support is selected from the group consisting of abead, a particulate, a membrane, a blended article, a graft copolymericarticle, a woven web, a nonwoven web, a solid plastic article having asurface comprising azlactone moieties, and combinations thereof.
 3. Themethod of claim 1, wherein the biologically active substance is selectedfrom the group consisting of antibodies, lectins, proteins, antigens,avidin, and combinations thereof.
 4. The method of claim 1, wherein thebiologically active substance directly interacts with the whole cells.5. The method of claim 1, wherein the biologically active substanceindirectly interacts with the whole cells through a second, intermediarybiologically active substance that is bifunctional to both the wholecells and the azlactone-functional support.
 6. The method of claim 1,wherein the azlactone-functional support is prepared by processesselected from the group consisting of suspension polymerizationprocesses and dispersion polymerization processes.
 7. The method ofclaim 6, wherein the aziactone-functional support is prepared from2-alkenyl azlactone monomers and, optionally, comonomers andcrosslinkers.
 8. The method of claim 2, wherein the solid plasticarticle is a microtitration well, a microtitration plate, a petri dish,medical tubing, a test tube, a centrifuge tube, a beaker, a cuvette, ora body implant.
 9. The method of claim 1, wherein the optional step (f)is used for further biological processing of the whole cells.
 10. Themethod of claim 1, wherein the mixture is selected from the groupconsisting of bone marrow and peripheral blood.
 11. A purified wholecell population produced by the method of claim
 1. 12. An interactedsupport, comprising: (a) an azlactone-functional support, (b) abiologically active substance covalently coupled to the support, and (c)a whole cell interacting with said substance.
 13. The support of claim12, wherein the wherein the azlactone-functional support is selectedfrom the group consisting of a bead, a particulate, a membrane, ablended article, a graft copolymeric article, a woven web, a nonwovenweb, a solid plastic article having a surface comprising azlactonemoieties, and combinations thereof.
 14. The support of claim 12, whereinthe biologically active substance is selected from the group consistingof antibodies, lectins, proteins, antigens, avidin, and combinationsthereof.
 15. The support of claim 12, wherein the biologically activesubstance indirectly interacts with the whole cells through a second,intermediary biologically active substance that is bifunctional to boththe whole cells and the azlactone-functional support.
 16. The support ofclaim 13, wherein the solid plastic article is a microtitration well, amicrotitration plate, a petri dish, medical tubing, a test tube, acentrifuge tube, a beaker, a cuvette, or a body implant.
 17. The supportof claim 12, wherein the aziactone-functional support prior to covalentcoupling with the biologically active substance has at least oneazlactone-functional group of a formula:

wherein: R¹ and R² independently can be an alkyl group having 1 to 14carbon atoms, a cycloalkyl group having 3 to 14 carbon atoms, an arylgroup having 5 to 12 ring atoms, an arenyl group having 6 to 26 carbonatoms and 0 to 3 S, N, and nonperoxidic 0 heteroatoms, or R¹ and R²taken together with the carbon to which they are joined can form acarbocyclic ring containing 4 to 12 ring atoms, and n is an integer 0 or1.